Polypeptides having antiviral activity and methods for use thereof

ABSTRACT

A polypeptide is provided that comprises an actinohivin variant polypeptide having an amino acid sequence selected from SEQ ID NOS: 4-12. The polypeptide can be provided as part of a fusion protein that includes the actinohivin variant polypeptide and either a fragment crystallizable domain of an antibody (Fc), a fragment antigen-binding domain of an antibody (Fab), or a single chain variable fragment of an antibody (scFv). Isolated nucleic acid molecules encoding the polypeptides are also provided along with vectors and plant cells capable of expressing the polypeptides. Methods of treating an infection of a subject by an enveloped virus are further provided and include administering an effective amount of the polypeptides to a subject.

GOVERNMENT INTEREST

This invention was made with government support under grant numberR21-AI 088585 provided by the National Institutes of Health and grantnumber W81XWH-09-2-0022 provided by the U.S. Army. The government hascertain rights in this invention.

RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application Ser.No. 61/362,227, filed Jul. 7, 2010, and U.S. Provisional ApplicationSer. No. 61/368,343, filed Jul. 28, 2010, the entire disclosures ofwhich are incorporated herein by this reference.

TECHNICAL FIELD

The presently-disclosed subject matter relates to polypeptides havingantiviral activity. In particular, the presently-disclosed subjectmatter relates to polypeptides comprising an actinohivin (AH)polypeptide or an actinohivin variant (AH_(var)) polypeptide and havingantiviral activity.

BACKGROUND

For a number of years, enveloped virus infections, including humanimmunodeficiency virus (HIV) infections, have posed serious globalhealth concerns. Indeed, millions of new HIV infections are reportedevery year worldwide, with a majority of those new infections occurringmainly in developing regions where the availability of antiretroviraldrug therapies is extremely limited, and, consequently, where AcquiredImmunodeficiency Syndrome (AIDS) is among the leading causes of death[1]. The majority of infections are established via heterosexualtransmission and condom use is currently the only available means todirectly block this route of infection. As such, there is an urgent needfor a woman-controlled, safe, effective, and inexpensive topicalmicrobicide, at least until prophylaxis through vaccination becomesglobally available [2, 3].

Current candidate microbicides include chemical and physical agents aswell as biologicals, such as virion-inactivating agents, entry/fusioninhibitors, reverse transcriptase inhibitors, and a number of others. Atthis point in time, however, it is not known which type of anti-HIVagents will prove to be the most effective topical microbicides and,indeed, the blocking of HIV-1 mucosal transmission may requirecombinations of multiple agents [4, 5]. Therefore, to broaden theoptions for different combinations in HIV-1 microbicide development, ithas been recognized that it is important to expand the candidateportfolio in each category of possible microbicide components.

In this regard, it has recently been further recognized that envelope(Env) glycans may constitute an attractive target for entry/fusioninhibitor-based microbicide development as Env glycans play criticalroles in broad aspects of the viral life cycle ranging from Env foldingin host cells to viral transmission and immune escape [7]. Indeed, ithas been observed that the envelope (Env) gp120 is heavily glycosylatedwith N-linked glycans (NLGs), and generally accounts for more than halfof the protein's molecular mass [6], with high-mannose-type glycans(HMGs) representing the major class.

Along these lines, lectins have attracted considerable attention in thesearch for Env glycan-targeting microbicide candidates as variousnaturally-occurring lectins have been shown to possess anti-HIVactivities. Examples include algae-derived cyanovirin-N(CV-N) andgriffithsin (GRFT), as well as plant-derived concanavalin A (Con A) andsnowdrop lectin, among others [7]. More recently, a Jacalin-relatedlectin isolated from the banana fruit was shown to potently inhibitHIV-1 entry into target cells [8]. Although conceptually not a lectin,the human monoclonal antibody (mAb) 2G12 has also been shown tospecifically bind to gp120 HMGs and thereby neutralize a wide spectrumof HIV-1 strains; thus, it has also been counted among the very fewbroadly neutralizing mAbs isolated to date [9,10].

One particular lectin that has recently been noted as having anti-HIVactivity is actinohivin (AH). AH was first isolated from theactinomycete strain Longispora albida K97-0003^(T) based on theinhibitory activity that was displayed in a syncytium formation assay[11]. Recent crystallographic analysis has revealed that AH is amonomeric protein and possesses three carbohydrate-binding (i.e.,sugar-binding) domains [12]. Unlike several other knownmonosaccharide-specific anti-HIV lectins such as GRFT, Con A, andGalanthus nivalis agglutinin [7], however, AH specifically recognizes acluster of multiple HMGs via a collaborative action among the protein'sthree sugar-binding sites [12, 13]. Because clustering HMGs is a uniquefeature of Env glycans and is not usually found on host human proteins[14], AH has been hypothesized to be a better anti-HIV-1 lectin withgreater specificity to the virus. To date, however, and despite thedetailed studies of AH's carbohydrate-binding specificity [12, 13, 15],limited investigation has been undertaken with respect to the protein'spotential anti-HIV activity and with respect to how the protein may bemanipulated to produce commercially relevant amounts and/or to provideincreased anti-HIV activity.

SUMMARY

The presently-disclosed subject matter meets some or all of theabove-identified needs, as will become evident to those of ordinaryskill in the art after a study of information provided in this document.This summary describes several embodiments of the presently-disclosedsubject matter, and in many cases lists variations and permutations ofthese embodiments. This summary is merely exemplary of the numerous andvaried embodiments. Mention of one or more representative features of agiven embodiment is likewise exemplary. Such an embodiment can typicallyexist with or without the feature(s) mentioned; likewise, those featurescan be applied to other embodiments of the presently-disclosed subjectmatter, whether listed in this summary or not. To avoid excessiverepetition, this summary does not list or suggest all possiblecombinations of such features.

The presently-disclosed subject matter relates to polypeptidescomprising an actinohivin (AH) polypeptide or an actinohivin variant(AH_(var)) polypeptide, and having antiviral activity. In someembodiments of the presently-disclosed subject matter, a polypeptide isprovided that comprises an AH_(var) polypeptide having an amino acidsequence selected from SEQ ID NOS: 4-12. In some embodiments, theAH_(var) polypeptide comprises the sequence of SEQ ID NO: 4. In someembodiments, the AH_(var) polypeptide further comprises and endomembranesystem (EMS)-targeting signal peptide, such as that provided in SEQ IDNO: 15.

In some embodiments of the presently-disclosed polypeptides, apolypeptide is provided in which the AH_(var) polypeptide is provided aspart of a fusion protein. For example, in some embodiments, an AH_(var)polypeptide is provided along with a second polypeptide selected from: afragment crystallizable domain of an antibody (Fc); a fragmentantigen-binding domain of an antibody (Fab); or a single chain variablefragment of an antibody (scFv), such that the AH_(var) polypeptide andthe second polypeptide form a fusion protein. In some embodiments, theAH_(var) polypeptide and the second polypeptide are connected via apeptide linker, such as that provided in SEQ ID NO: 3. In certain ofthese embodiments, the antibody domains used in these fusion proteinsare derived from a monoclonal antibody.

In further embodiments of the presently-disclosed subject matter, fusionproteins are provided that comprise a native actinohivin polypeptide.For example, in some embodiments, a fusion polypeptide is provided thatcomprises an actinohivin polypeptide; and a second polypeptide selectedfrom: a fragment antigen-binding domain of an antibody (Fab); and asingle chain variable fragment of an antibody (scFv).

Further provided, in some embodiments of the presently-disclosed subjectmatter, are isolated nucleic acid molecules. In some embodiments, anisolated nucleic acid molecule is provided that comprises a sequencethat encodes an actinohivin variant polypeptide having an amino acidsequence selected from SEQ ID NOS: 4-12. In some embodiments, thenucleic acid molecule comprises the amino acid sequence of SEQ ID NO: 4.In some embodiments, the nucleic acid molecule comprises the nucleicacid sequence of SEQ ID NO: 3.

In some embodiments, the isolated nucleic acid molecules of thepresently-disclosed subject matter are provided as part of a vectorthat, in certain embodiments, includes an expression control sequenceoperably linked to the isolated nucleic acid molecules. In someembodiments, a plant cell is provided that has been transfected withsuch a vector to thereby provide a plant cell, or a progeny thereof,that expresses the actinohivin variant polypeptide. In some embodiments,the plant cell is a Nicotiana benthamiana plant cell.

Still further provided, in some embodiments of the present disclosedsubject matter, are pharmaceutical compositions. In some embodiments, apharmaceutical composition is provided that comprises an AH_(var)polypeptide having an amino acid sequence selected from SEQ ID NOS: 4-12and a pharmaceutically-acceptable vehicle, carrier, or excipient.

In yet further embodiments of the presently-disclosed subject matter,therapeutic and/or prophylactic methods are provided that make use ofthe polypeptides described herein. In some embodiments, a method oftreating an infection of a subject by an enveloped virus is provided. Insome embodiments, a method of treating an infection of a subject by anenveloped virus is provided that includes the step of administering tothe subject an effective amount of an AH_(var) polypeptide having anamino acid sequence selected from SEQ ID NOS: 4-12. In some embodiments,the enveloped virus is a human immunodeficiency virus (HIV), aninfluenza virus, a hanta virus, a hepatitis C virus, a herpes virus, asevere acute respiratory syndrome coronavirus (SARS-CoV), ametapneumovirus, a henipavirus, a flavivirus, or a hemorrhagic fevervirus. In some embodiments, administering the AH_(var) polypeptide tothe subject comprises topically administering the AH_(var) polypeptide.

Further features and advantages of the presently-disclosed subjectmatter will become evident to those of ordinary skill in the art after astudy of the description, figures, and non-limiting examples in thisdocument.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1J includes amino acid sequences of a native actinohivin (AH)polypeptide (FIG. 1A, SEQ ID NO: 2) and of nine actinohivin variant(AH_(var1-9)) polypeptides (FIGS. 1B-1J; SEQ ID NOS: 4-12, respectively)of the presently-disclosed subject matter, where thecarbohydrate-binding domains of the polypeptides are underlined, whereinnon-underlined residues comprise hinge or terminal extension regions ofthe polypeptides, and where the amino acid residues that are changedrelative to the native AH amino acid sequence are shown in bold anditalic font;

FIG. 2 is an image of a gel showing the SDS-PAGE analysis of the amountsof native AH polypeptides (Native AH) and AH_(var1) polypeptides (MutantAH) produced in Nicotiana benthamiana plants;

FIG. 3 is a graph showing the results of an Env-CD₄ syncytium formationassay used to measure the anti-human immunodeficiency virus (HIV)activity of an AH_(var1) polypeptide, where the percent of syncytiumformation is plotted against the concentrations of the polypeptides usedin the assay, and where the results obtained with particularconcentrations of the AH_(var1) polypeptide (Plant mAH) are plottedalong with the results obtained using the same concentrations of nativeAH (Standard AH);

FIG. 4 is a schematic representation of an AH_(var)-Fc fusion protein ofthe presently-disclosed subject matter;

FIG. 5 includes images of gels showing the SDS-PAGE analysis of anAH_(var1) polypeptide produced in Nicotiana benthamiana plants underreducing condition and non-reducing conditions at 8 days post vectorinoculation (dpi); and

FIG. 6 is a graph showing the results of an in vitro HIV neutralizationassay where the percent of HIV neutralization is plotted as a functionof the concentration of AH_(var)-Fc fusion polypeptide or actinohivin(AH) polypeptide used in the assay.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: 1 is a nucleic acid sequence encoding a native actinohivin(AH) polypeptide;

SEQ ID NO: 2 is an amino acid sequence of a native AH polypeptide;

SEQ ID NO: 3 is a nucleic acid sequence encoding a first exemplaryactinohivin variant (AH_(var1)) polypeptide made in accordance with thepresently-disclosed subject matter;

SEQ ID NO: 4 is an amino acid sequence of the AH_(var1) polypeptide;

SEQ ID NO: 5 is an amino acid sequence of a second exemplary actinohivinvariant (AH_(var2)) polypeptide made in accordance with thepresently-disclosed subject matter;

SEQ ID NO: 6 is an amino acid sequence of a third exemplary actinohivinvariant (AH_(var3)) polypeptide made in accordance with thepresently-disclosed subject matter;

SEQ ID NO: 7 is an amino acid sequence of a fourth exemplary actinohivinvariant (AH_(var4)) polypeptide made in accordance with thepresently-disclosed subject matter;

SEQ ID NO: 8 is an amino acid sequence of a fifth exemplary actinohivinvariant (AH_(var5)) polypeptide made in accordance with thepresently-disclosed subject matter;

SEQ ID NO: 9 is an amino acid sequence of a sixth exemplary actinohivinvariant (AH_(var6)) polypeptide made in accordance with thepresently-disclosed subject matter;

SEQ ID NO: 10 is an amino acid sequence of a seventh exemplaryactinohivin variant (AH_(var7)) polypeptide made in accordance with thepresently-disclosed subject matter;

SEQ ID NO: 11 is an amino acid sequence of an eighth exemplaryactinohivin variant (AH_(var8)) polypeptide made in accordance with thepresently-disclosed subject matter; and SEQ ID NO: 12 is an amino acidsequence of a ninth exemplary actinohivin variant (AH_(var9))polypeptide made in accordance with the presently-disclosed subjectmatter;

SEQ ID NO: 13 is an amino acid sequence of a peptide linker;

SEQ ID NO: 14 is a nucleic acid sequence encoding an endomembrane system(EMS)-targeting signal peptide; and

SEQ ID NO: 15 is an amino acid sequence of an endomembrane system(EMS)-targeting signal peptide.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

The details of one or more embodiments of the presently-disclosedsubject matter are set forth in this document. Modifications toembodiments described in this document, and other embodiments, will beevident to those of ordinary skill in the art after a study of theinformation provided in this document. The information provided in thisdocument, and particularly the specific details of the describedexemplary embodiments, is provided primarily for clearness ofunderstanding and no unnecessary limitations are to be understoodtherefrom. In case of conflict, the specification of this document,including definitions, will control.

As will be recognized by those skilled in the art, certain polypeptidesand nucleotides described herein can be found in publicly-accessibledatabases, such as in the GENBANK® and SWISSPROT databases. Thesequences contained in such databases are incorporated herein byreference as are equivalent and related sequences present in GENBANK®,SWISSPROT, or other public databases. Also expressly incorporated hereinby reference are all annotations present in the GENBANK® and SWISSPROTdatabases associated with the polypeptides and nucleotides describedherein. Unless otherwise indicated or apparent, the references to theGENBANK® database and the SWISSPROT database are references to the mostrecent version of the database as of the filing date of thisApplication.

While the following terms used herein are believed to be well understoodby one of ordinary skill in the art, definitions are set forth tofacilitate explanation of the presently-disclosed subject matter. Unlessdefined otherwise, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which the presently-disclosed subject matter belongs. Althoughany methods, devices, and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of thepresently-disclosed subject matter, representative methods, devices, andmaterials are now described.

Following long-standing patent law convention, the terms “a”, “an”, and“the” refer to “one or more” when used in this application, includingthe claims. Thus, for example, reference to “a cell” includes aplurality of such cells, and so forth.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as reaction conditions, and so forth usedin the specification and claims are to be understood as being modifiedin all instances by the term “about.” Accordingly, unless indicated tothe contrary, the numerical parameters set forth in this specificationand claims are approximations that can vary depending upon the desiredproperties sought to be obtained by the presently-disclosed subjectmatter.

As used herein, the term “about,” when referring to a value or to anamount of mass, weight, time, volume, concentration or percentage ismeant to encompass variations of in some embodiments ±20%, in someembodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, insome embodiments ±0.5%, and in some embodiments ±0.1% from the specifiedamount, as such variations are appropriate to perform the disclosedmethods.

Actinohivin (AH) is an actinomycete-derived lectin that is produced as amonomeric protein having three carbohydrate-binding sites or domains[12]. AH has been found to specifically bind to high-mannose-typeglycans (HMGs) found on the envelope (Env) of a number of envelopedviruses and, more specifically, has been found to specifically bind to acluster of HMGs uniquely found on the HIV-1 Env such that AH is capableof eliciting nanomolar antiviral activity against multiple HIV strains.Analyses have revealed that AH has a high safety profile in humanperipheral blood mononuclear cells (PBMCs) and in a rabbit vaginalirritation assay, and it has been further demonstrated that atranslational AH-AH fusion protein exhibits stronger and broaderanti-HIV-1 activity than the original monomeric protein, potentially dueto stronger binding to the target as a result of high avidity and/orbetter cross-linking of Env [61]. It has now been discovered thatmodifications can be made in the AH amino acid sequence to provideactinohivin variant (AH_(var)) polypeptides that can be provided alone(e.g., as monomers or dimers) or in the form of a fusion proteins, andthat have surprisingly superior properties relative to a native AHpolypeptide. For example, in some embodiments, polypeptides comprisingan AH_(var) have reduced hydrophobicity and surface charge properties(e.g., isoelectric point [pI]) as compared to native AH polypeptide,which allows for the AH_(var) polypeptides to be expressed at higherlevels in plant cells and also allows the AH_(var) polypeptides to bemore readily extracted and purified. For another example, in someembodiments, polypeptides comprising AH_(var) have improved stability ascompared to native AH polypeptides.

In some embodiments, the presently-disclosed subject matter thusprovides isolated polypeptides that comprise an AH_(var) polypeptide. Insome embodiments, an AH, polypeptide is provided that has an amino acidsequence selected from SEQ ID NOS: 4-12. In some embodiments, theAH_(var) polypeptide comprises the amino acid sequence of SEQ ID NO: 4.

The term “isolated,” when used in the context of an isolatedpolypeptide, or an isolated nucleic acid, is a polypeptide, or nucleicacid, that, by the hand of man, exists apart from its native environmentand is therefore not a product of nature. An isolated polypeptide ornucleic acid can exist in a purified form or can exist in a non-nativeenvironment such as, for example, in a transgenic host cell.

The term “native”, when used with reference to a polypeptide, refers toa polypeptide that is encoded by a gene that is naturally present in thegenome of an untransformed cell. In this regard, the term “gene” is usedbroadly to refer to any segment of DNA associated with a biologicalfunction. Thus, genes include, but are not limited to, coding sequencesand/or the regulatory sequences required for their expression. Genes canalso include non-expressed DNA segments that, for example, formrecognition sequences for a polypeptide. Genes can be obtained from avariety of sources, including cloning from a source of interest orsynthesizing from known or predicted sequence information, and caninclude sequences designed to have desired parameters.

The terms “polypeptide”, “protein”, and “peptide”, which are usedinterchangeably herein, refer to a polymer of the 20 protein aminoacids, or amino acid analogs, regardless of its size or function.Although “protein” is often used in reference to relatively largepolypeptides, and “peptide” is often used in reference to smallpolypeptides, usage of these terms in the art overlaps and varies. Theterm “polypeptide” as used herein refers to peptides, polypeptides, andproteins, unless otherwise noted. The terms “protein”, “polypeptide” and“peptide” are used interchangeably herein when referring to a geneproduct. Thus, exemplary polypeptides include gene products, nativeproteins, homologs, orthologs, paralogs, fragments and otherequivalents, variants, and analogs of the foregoing.

The terms “polypeptide fragment” or “fragment”, when used in referenceto a reference polypeptide, refers to a polypeptide in which amino acidresidues are deleted as compared to the reference polypeptide itself,but where the remaining amino acid sequence is usually identical to thecorresponding positions in the reference polypeptide. Such deletions canoccur at the amino-terminus or carboxy-terminus of the referencepolypeptide, or alternatively both. A fragment can also be a “functionalfragment,” in which case the fragment retains some or all of theactivity of the reference polypeptide as described herein. For example,a functional fragment of an AH polypeptide can retain some or all of theability of the reference polypeptide to bind carbohydrates, such asHMGs.

The terms “modified amino acid”, “modified polypeptide”, and “variant”refer to an amino acid sequence that is different from the referencepolypeptide by one or more amino acids, e.g., one or more amino acidsubstitutions. A variant of a reference polypeptide also refers to avariant of a fragment of the reference polypeptide, for example, afragment wherein one or more amino acid substitutions have been maderelative to the reference polypeptide. A variant can also be a“functional variant,” in which the variant retains some or all of theactivity of the reference protein as described herein. For example, insome embodiments, an AH_(var) polypeptide can retain some or all of theability of a native AH polypeptide to bind HMGs.

The term functional variant also includes a functional variant of afunctional fragment of a reference polypeptide. The term functionalvariant further includes conservatively substituted variants. The term“conservatively substituted variant” refers to a peptide comprising anamino acid residue sequence that differs from a reference peptide by oneor more conservative amino acid substitutions, and maintains some or allof the activity of the reference peptide as described herein. A“conservative amino acid substitution” is a substitution of an aminoacid residue with a functionally similar residue. Examples ofconservative substitutions include the substitution of one non-polar(hydrophobic) residue such as isoleucine, valine, leucine or methioninefor another; the substitution of one charged or polar (hydrophilic)residue for another such as between arginine and lysine, betweenglutamine and asparagine, between threonine and serine; the substitutionof one basic residue such as lysine or arginine for another; or thesubstitution of one acidic residue, such as aspartic acid or glutamicacid for another; or the substitution of one aromatic residue, such asphenylalanine, tyrosine, or tryptophan for another. The phrase“conservatively substituted variant” also includes peptides wherein aresidue is replaced with a chemically-derivatized residue, provided thatthe resulting peptide maintains some or all of the activity of thereference peptide as described herein.

As noted, in some embodiments, the polypeptides of thepresently-disclosed subject matter include more than one AH or AH_(var)polypeptide. For example, in some embodiments, the polypeptide caninclude two AH_(var) polypeptides such that the AH_(var) polypeptidesare provided as a dimer. As another example, in some embodiments, thepolypeptides can include an AH_(var) polypeptide and an AH polypeptide.

In some embodiments of the presently-disclosed polypeptides, the AHand/or AH_(var) polypeptides can be provided as part of a fusion proteinsuch that a protein can be provided that confers antibody-dependentcell-mediated cytotoxicity, complement-dependent cytotoxicity,antibody-dependent cellular phagocytosis, antibody-dependentcell-mediated virus inhibition, and/or a longer serum half life. In someembodiments, a fusion protein is provided that includes: an AH_(var)polypeptide having an amino acid sequence selected from SEQ ID NOS:4-12; and a second polypeptide selected from a fragment crystallizabledomain of an antibody (Fc); a fragment antigen-binding domain of anantibody (Fab); and a single chain variable fragment of an antibody(scFv), wherein the AH_(var) polypeptide and the second polypeptidetogether comprise the fusion protein. For example, in some embodiments,the fusion protein includes an AH_(var) polypeptide and the fragmentcrystallizble (Fc) domain of a human monoclonal immunoglobulin (Ig) G(AH_(var)-Fc). As another example, in some embodiments, a fusion proteincan be provided that includes an AH_(var) polypeptide and the fragmentantigen binding (Fab) domain of a human monoclonal antibody. As yetanother example, in some embodiments, a fusion protein can be providedthat includes an AH_(var) polypeptide and the single chain variablefragment (scFv) domain of an antibody.

In some embodiments of the fusion proteins described herein, a fusionprotein is provided that includes a native AH polypeptide and a secondpolypeptide selected from a Fab fragment and a scFv fragment.

As used herein, the phrase “fusion polypeptide” or “fusion protein” isused to refer to a polypeptide made up of two or more amino acidsequences representing peptides or polypeptides from different sources.In some embodiments, a peptide linker sequence may be employed toseparate the first and the second polypeptides by a distance sufficientto ensure that each polypeptide folds into its secondary and tertiarystructures. Such a peptide linker sequence can be incorporated into thefusion protein using standard techniques well known in the art, and canbe chosen based on: (1) the ability of the linker sequence to adopt aflexible extended conformation; (2) the ability of the linker sequenceto adopt a secondary structure that could interact with functionaldomains on the first and second polypeptides; and (3) the lack ofhydrophobic or charged residues that might react with the polypeptidefunctional domains. Preferred peptide linker sequences contain Gly, Asnand Ser residues. Other near neutral amino acids, such as Thr and Alamay also be used in the linker sequence. Amino acid sequences which maybe usefully employed as linkers include those disclosed in Maratea etal., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA83:8258-8562, 1986; U.S. Pat. No. 4,935,233 and U.S. Pat. No. 4,751,180.In some embodiments, the linker sequence can be from 1 to about 50 aminoacids in length. In some embodiments, the linker comprises an amino acidsequence of SEQ ID NO: 13.

Fusion proteins in accordance with the presently-disclosed subjectmatter can be produced by a variety of methods known to those ofordinary skill in the art. For example, in some embodiments, a fusionprotein comprising an AH_(var) polypeptide and the Fc region of humanimmunoglobulin (Ig)G can be produced by attaching the nucleic acidsequence encoding the hinge and the Fc region of human IgG to the 3′ endof an AH_(var) polypeptide coding sequence, such as that provided in SEQID NO: 3, via the use of a coding sequence for a flexible peptide linker(e.g., Gly-Gly-Gly-Ser; SEQ ID NO: 13). In some embodiments, theconstructed nucleic acid sequence is then inserted into an appropriatevector for expression of the fusion protein in a desired cell. In someembodiments, such as those where the fusion polypeptides are to beproduced in plants, a nucleic acid coding sequence for an endomembranesystem (EMS)-targeting signal peptide, such as what is provided in SEQID NOS: 14 and 15 as well as those derived from tobacco calreticulin, N.benthamiana chitinases, and others can further be attached to the 5′ endof the nucleic acid sequence encoding the AH_(var) polypeptide. Ofcourse, various other methods and signal sequences can also be used inaccordance with the production of the presently-disclosed AH_(var)polypeptides as would be understood by those of ordinary skill in theart.

As noted, in some embodiments of the fusion proteins described herein,the antibody fragments used in accordance with the presently-disclosedsubject matter are monoclonal antibody fragments. However, any antibodyor fragments thereof which effectively confer antibody-dependentcellular cytotoxicity, complement-dependent cytotoxicity, and/or longerserum half life to the AH or AH_(var) polypeptides described herein arewithin the scope of the presently-disclosed subject matter. Thisincludes by way of example, monoclonal antibodies, recombinantantibodies, chimeric antibodies, humanized antibodies, bispecificantibodies, catalytic antibodies, single chain antibodies, antibodiesfrom different species (e.g., mouse, goat, rabbit, human, rat, bovine,llama, etc.), anti-idiotypic antibodies, antibodies of different isotype(IgG, IgM, IgE, IgA, etc.), as well as fragments and derivatives thereof(e.g., (Fab)₂, Fab, Fv, Fab, 2(Fab), Fab′, (Fab)₂ fragments).

Further provided, in some embodiments of the presently-disclosed subjectmatter, are isolated nucleic acids encoding one or more of thepolypeptides described herein (i.e., an AH_(var) polypeptide). In someembodiments, an isolated nucleic acid molecule is provided thatcomprising a sequence encoding an AH_(var) polypeptide having an aminoacid sequence selected from SEQ ID NOS: 4-12. In some embodiments, anisolated nucleic acid molecule is provided that comprises the sequenceof SEQ ID NO: 3.

The term “nucleic acid” refers to deoxyribonucleotides orribonucleotides and polymers thereof in either single- ordouble-stranded form. Unless specifically limited, the term encompassesnucleic acids containing known analogues of natural nucleotides thathave similar binding properties as the reference nucleic acid and aremetabolized in a manner similar to naturally-occurring nucleotides.Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified or degenerate variantsthereof (e.g., degenerate codon substitutions) and complementarysequences, as well as the sequence explicitly indicated.

The term “degenerate variant” refers to a nucleic acid having a residuesequence that differs from a reference nucleic acid by one or moredegenerate codon substitutions. Degenerate codon substitutions can beachieved by generating sequences in which the third position of one ormore selected (or all) codons is substituted with mixed base and/ordeoxyino sine residues (Batzer, et al. 1991; Ohtsuka, et al. 1985;Rossolini, et al. 1994).

In some embodiments, an isolated nucleic acid sequence is provided thatselectively hybridizes to the sequence of SEQ ID NO: 3. The term“selectively hybridize” as used herein refers to the ability of anucleic acid sequence to hybridize to a target polynucleotide (e.g., apolynucleotide of SEQ ID NO: 3) with specificity. Thus, the nucleic acidsequence comprises a polynucleotide sequence that is complementary, oressentially complementary, to at least a portion of the targetpolynucleotide sequence. For example, in some embodiments, the nucleicacid sequence that selectively hybridizes to the sequence of SEQ ID NO:3 is complementary to the sequence of SEQ ID NO: 3. Nucleic acidsequences which are “complementary” are those which are base-pairingaccording to the standard Watson-Crick complementarity rules. As usedherein, the term “complementary sequences” means nucleic acid sequenceswhich are substantially complementary, as can be assessed by the samenucleotide comparison set forth above, or as defined as being capable ofhybridizing to the nucleic acid segment in question under relativelystringent conditions such as those described herein. A particularexample of a contemplated complementary nucleic acid segment is anantisense oligonucleotide. With regard to the nucleic acid sequencesdisclosed herein as selectively hybridizing to the sequence of SEQ IDNO: 3, the hybridizing nucleic acid sequence need not necessarily becompletely complementary to the nucleic acid of SEQ ID NO: 3 along theentire length of the target polynucleotide so long as the hybridizingnucleic acid sequence can bind the nucleic acid of SEQ ID NO: 3 withspecificity. In some embodiments, the nucleic acid sequences thatselectively hybridize to the sequence of SEQ ID NO: 3 are about 80%,about 85%, about 90%, about 95%, about 98%, or about 100% complementaryto the sequence of SEQ ID NO: 3.

Nucleic acid hybridization will be affected by such conditions as saltconcentration, temperature, or organic solvents, in addition to the basecomposition, length of the complementary strands, and the number ofnucleotide base mismatches between the hybridizing nucleic acids, aswill be readily appreciated by those skilled in the art. Stringenttemperature conditions will generally include temperatures in excess of30° C., typically in excess of 37° C., and preferably in excess of 45°C. Stringent salt conditions will ordinarily be less than 1,000 mM,typically less than 500 mM, and preferably less than 200 mM. Forexample, in some embodiments, nucleic acid hybridization can beperformed at 60° C. with 0.1× sodium citrate-sodium chloride (SSC) and0.1% sodium dodecyl sulfate (SDS). However, the combination ofparameters is much more important than the measure of any singleparameter. (See, e.g., Wetmur & Davidson, 1968). Determining appropriatehybridization conditions to identify and/or isolate sequences containinghigh levels of homology is well known in the art. (See, e.g., Sambrook,et al., 1989).

In some embodiments of the presently-disclosed subject matter, vectorsthat include one or more of the isolated nucleic acid sequencesdisclosed herein are provided. In some embodiments, a vector is providedthat includes an isolated nucleic acid comprising a sequence of SEQ IDNO: 3. In some embodiments, a vector is provided that includes anisolated nucleic acid sequence that encodes an AH_(var) polypeptidehaving an amino acid sequence selected from SEQ ID NOS: 4-12. In someembodiments, a vector is provided that includes an isolated nucleic acidsequence that encodes a polypeptide comprising an amino acid sequence ofSEQ ID NO: 4.

The term “vector” is used herein to refer to any vehicle that is capableof transferring a nucleic acid sequence into a cell. For example,vectors which may be used in accordance with the presently-disclosedsubject matter include, but are not limited to, plasmids, cosmids,bacteriophages, or viruses, which can be transformed by the introductionof a nucleic acid sequence of the presently-disclosed subject matter.Such vectors are well known to those of ordinary skill in the art. Insome embodiments, the vectors of the presently-disclosed subject matterare plasmids. In other embodiment of the presently-disclosed subjectmatter, the vectors of the presently-disclosed subject matter areviruses, such as a tobamoviruses (e.g., tobacco mosaic virus, turnipvein-clearing virus, tomato mosaic virus, etc.), cowpea mosaic virus,potato virus X, geminiviruses, among others, as such viruses have beenfound to be particularly useful for introducing a nucleic acid sequenceof the presently-disclosed subject matter into a plant cell and forsubsequently expressing the protein encoded by the nucleic acid in theplant cell.

In some embodiments, the isolated nucleic acid included in the vector isoperably linked to an expression control sequence. The terms “associatedwith,” “operably linked,” and “operatively linked” refer to two nucleicacid sequences that are related physically or functionally. For example,a promoter or regulatory DNA sequence is said to be “associated with” aDNA sequence that encodes an RNA or a polypeptide if the two sequencesare operatively linked, or situated such that the regulator DNA sequencewill affect the expression level of the coding or structural DNAsequence.

The term “expression control sequence” refers to a nucleic acid moleculecapable of directing expression of a particular nucleotide sequence inan appropriate host cell, comprising a promoter operatively linked tothe nucleotide sequence of interest which is operatively linked totermination signals. It also typically comprises sequences required forproper translation of the nucleotide sequence. The coding region usuallyencodes a polypeptide of interest but can also encode a functional RNAof interest, for example antisense RNA or a non-translated RNA, in thesense or antisense direction. The expression control sequence comprisingthe nucleotide sequence of interest can be chimeric, meaning that atleast one of its components is heterologous with respect to at least oneof its other components. The expression control sequence can also be onethat is naturally occurring but has been obtained in a recombinant formuseful for heterologous expression.

The presently-disclosed subject matter also provides transgenic plantcells or plants that have been transfected with one or more of thevectors disclosed herein. As used herein, the term “plant cell” isunderstood to mean any cell derived from a monocotyledonous or adicotyledonous plant and capable of constituting undifferentiatedtissues such as calli, differentiated tissues such as embryos, portionsof monocotyledonous or dicotyledonous plants, monocotyledonous ordicotyledonous plants or seed. The term “plant” is understood to meanany differentiated multi-cellular organism capable of photosynthesis,including monocotyledons and dicotyledons. In some embodiments, theplant cell can be a tobacco plant cell, such as a Nicotiana benthamianaplant cell.

The terms “transformed,” “transgenic,” and “recombinant” are used hereinto refer to a cell of a host organism, such as a plant, into which aheterologous nucleic acid molecule has been introduced. The nucleic acidmolecule can be stably integrated into the genome of the cell or thenucleic acid molecule can also be present as an extrachromosomalmolecule. Such an extrachromosomal molecule can be auto-replicating.Transformed cells, tissues, or subjects are understood to encompass notonly the end product of a transformation process, but also transgenicprogeny thereof.

The terms “heterologous,” “recombinant,” and “exogenous,” when usedherein to refer to a nucleic acid sequence (e.g., a DNA sequence) or agene, refer to a sequence that originates from a source foreign to theparticular host cell or, if from the same source, is modified from itsoriginal form. Thus, a heterologous gene in a host cell includes a genethat is endogenous to the particular host cell but has been modifiedthrough, for example, the use of site-directed mutagenesis or otherrecombinant techniques. The terms also include non-naturally occurringmultiple copies of a naturally occurring DNA sequence. Thus, the termsrefer to a DNA segment that is foreign or heterologous to the cell, orhomologous to the cell but in a position or form within the host cell inwhich the element is not ordinarily found. Similarly, when used in thecontext of a polypeptide or amino acid sequence, an exogenouspolypeptide or amino acid sequence is a polypeptide or amino acidsequence that originates from a source foreign to the particular hostcell or, if from the same source, is modified from its original form.Thus, exogenous DNA segments can be expressed to yield exogenouspolypeptides.

Introduction of a nucleic acid (e.g., a nucleic acid incorporated intoan appropriate vector) of the presently-disclosed subject matter into aplant cell can be performed by a variety of methods known to those ofordinary skill in the art including, but not limited to, insertion of anucleic acid sequence of interest into an Agrobacterium rhizogenes Ri orAgrobacterium tumefaciens Ti plasmid, microinjection, electroporation,or direct precipitation. By way of providing an example, in someembodiments, transient expression of a nucleic acid sequence or gene ofinterest can be performed by agro-infiltration methods. In this regard,a suspension of Agrobacterium tumefaciens containing a nucleic acidsequence or gene of interest (e.g., due to electroporation) can be grownin culture and then a suspension of the bacterial culture can beinfiltrated into the plant by applying a vacuum. Once inside the leaf,the Agrobacterium transforms the gene of interest to a portion of theplant cells where the gene is then expressed. For additional informationand guidance regarding the expression of a nucleic acid sequence ofinterest in a plant cell, see, e.g., Matoba N, et al. Methods Mol. Biol.2011; 701:199-219; and, Matoba N, et al. PLoS One. 2010 Jun. 15;5(6):e11143, each of which are incorporated herein by this reference.

For further guidance regarding methods of transforming and producingtransgenic plant cells, see U.S. Pat. Nos. 4,459,355; 4,536,475;5,464,763; 5,177,010; 5,187,073; 4,945,050; 5,036,006; 5,100,792;5,371,014; 5,478,744; 5,179,022; 5,565,346; 5,484,956; 5,508,468;5,538,877; 5,554,798; 5,489,520; 5,510,318; 5,204,253; 5,405,765; EPNos. 267,159; 604,662; 672,752; 442,174; 486,233; 486,234; 539,563;674,725; and, International Patent Application Publication Nos. WO91/02071 and WO 95/06128, each of which is incorporated herein by thisreference.

Still further provided, in some embodiments of the presently-disclosedsubject matter are pharmaceutical compositions comprising a polypeptideof the presently-disclosed subject matter and apharmaceutically-acceptable vehicle, carrier, or excipient. For example,in some embodiments, a pharmaceutical composition is provided thatcomprises: a polypeptide comprising an AH_(var) polypeptide having anamino acid sequence selected from SEQ ID NOS: 4-12; and apharmaceutically-acceptable vehicle, carrier, or excipient. As anotherexample, in some embodiments, a pharmaceutical composition is providedthat comprises a fusion protein of the presently-disclosed subjectmatter (e.g., a fusion protein comprising an AH_(var) polypeptide havingan amino acid sequence selected from SEQ ID NOS: 4-12, and a Fc, Fab, orscFV fragment of an antibody) and a pharmaceutically-acceptable vehicle,carrier, or excipient.

For example, solid formulations of the compositions for oraladministration can contain suitable carriers or excipients, such as cornstarch, gelatin, lactose, acacia, sucrose, microcrystalline cellulose,kaolin, mannitol, dicalcium phosphate, calcium carbonate, sodiumchloride, or alginic acid. Disintegrators that can be used include, butare not limited to, microcrystalline cellulose, corn starch, sodiumstarch glycolate, and alginic acid. Tablet binders that can be usedinclude acacia, methylcellulose, sodium carboxymethylcellulose,polyvinylpyrrolidone (POVIDONE™), hydroxypropyl methylcellulose,sucrose, starch, and ethylcellulose. Lubricants that can be used includemagnesium stearates, stearic acid, silicone fluid, talc, waxes, oils,and colloidal silica. Further, the solid formulations can be uncoated orthey can be coated by known techniques to delay disintegration andabsorption in the gastrointestinal tract and thereby provide asustained/extended action over a longer period of time. For example,glyceryl monostearate or glyceryl distearate can be employed to providea sustained-/extended-release formulation. Numerous techniques forformulating sustained release preparations are known to those ofordinary skill in the art and can be used in accordance with the presentinvention, including the techniques described in the followingreferences: U.S. Pat. Nos. 4,891,223; 6,004,582; 5,397,574; 5,419,917;5,458,005; 5,458,887; 5,458,888; 5,472,708; 6,106,862; 6,103,263;6,099,862; 6,099,859; 6,096,340; 6,077,541; 5,916,595; 5,837,379;5,834,023; 5,885,616; 5,456,921; 5,603,956; 5,512,297; 5,399,362;5,399,359; 5,399,358; 5,725,883; 5,773,025; 6,110,498; 5,952,004;5,912,013; 5,897,876; 5,824,638; 5,464,633; 5,422,123; and 4,839,177;and WO 98/47491, each of which is incorporated herein by this reference.

Furthermore, liquid formulations of the compositions for oraladministration can be prepared in water or other aqueous vehicles, andcan contain various suspending agents such as methylcellulose,alginates, tragacanth, pectin, kelgin, carrageenan, acacia,polyvinylpyrrolidone, and include solutions, emulsions, syrups, andelixirs containing, together with the active components of thecomposition, wetting agents, sweeteners, and coloring and flavoringagents.

Various liquid and powder formulations can also be prepared byconventional methods for inhalation into the lungs of the subject to betreated. For example, the compositions can be conveniently delivered inthe form of an aerosol spray presentation from pressurized packs or anebulizer, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas.Capsules and cartridges of, for example, gelatin for use in an inhaleror insufflator may be formulated containing a powder mix of the desiredcompound and a suitable powder base such as lactose or starch.

Injectable formulations of the compositions can contain various carrierssuch as vegetable oils, dimethylacetamide, dimethylformamide, ethyllactate, ethyl carbonate, isopropyl myristate, ethanol, polyols(glycerol, propylene glycol, liquid polyethylene glycol), and the like.For intravenous injections, water soluble versions of the compositionscan be administered by the drip method, whereby a formulation includinga pharmaceutical composition of the presently-disclosed subject matterand a physiologically-acceptable excipient is infused.Physiologically-acceptable excipients can include, for example, 5%dextrose, 0.9% saline, Ringer's solution or other suitable excipients.Intramuscular preparations, e.g., a sterile formulation of a suitablesoluble salt form of the compositions, can be dissolved and administeredin a pharmaceutical excipient such as Water-for-Injection, 0.9% saline,or 5% glucose solution. A suitable insoluble form of the compositionscan be prepared and administered as a suspension in an aqueous base or apharmaceutically-acceptable oil base, such as an ester of a long chainfatty acid, (e.g., ethyl oleate).

In addition to the formulations described above, the compositions of thepresently-disclosed subject matter can also be formulated as rectalcompositions, such as suppositories or retention enemas, e.g.,containing conventional suppository bases such as cocoa butter or otherglycerides. Further, the compositions can also be formulated as a depotpreparation by combining the compositions with suitable polymeric orhydrophobic materials (for example as an emulsion in an acceptable oil)or ion exchange resins, or as sparingly soluble derivatives, forexample, as a sparingly soluble salt.

In some embodiments of the present invention, the compositions of thepresent invention may be incorporated as part of a nanoparticle. A“nanoparticle” within the scope of the presently-disclosed subjectmatter is meant to include particles at the single molecule level aswell as those aggregates of particles that exhibit microscopicproperties. Methods of using and making a nanoparticle that incorporatesa compound of interest are known to those of ordinary skill in the artand can be found following references: U.S. Pat. Nos. 6,395,253,6,387,329, 6,383,500, 6,361,944, 6,350,515, 6,333,051, 6,323,989,6,316,029, 6,312,731, 6,306,610, 6,288,040, 6,272,262, 6,268,222,6,265,546, 6,262,129, 6,262,032, 6,248,724, 6,217,912, 6,217,901,6,217,864, 6,214,560, 6,187,559, 6,180,415, 6,159,445, 6,149,868,6,121,005, 6,086,881, 6,007,845, 6,002,817, 5,985,353, 5,981,467,5,962,566, 5,925,564, 5,904,936, 5,856,435, 5,792,751, 5,789,375,5,770,580, 5,756,264, 5,705,585, 5,702,727, and 5,686,113, each of whichis incorporated herein by this reference.

A topical formulation (e.g., a semi-solid ointment formulation) can alsobe provided and can contain a desired concentration of the activeingredient (e.g., a polypeptide of the presently-disclosed subjectmatter) in a carrier such as a pharmaceutical cream base. Variousformulations for topical use include drops, tinctures, lotions, creams,solutions, and ointments containing the active ingredient and varioussupports and vehicles. The optimal percentage of the therapeutic agentin each pharmaceutical formulation varies according to the formulationitself and the therapeutic effect desired in the specific pathologiesand correlated therapeutic.

In yet further embodiments of the presently-disclosed subject matter,methods of treating a viral infection are provided. In some embodiments,a method of treating an infection of a subject by an enveloped virus isprovided that includes administering an effective amount of apolypeptide of the presently-disclosed subject matter to the subject.

As used herein, the terms “treatment” or “treating” relate to anytreatment of an infection of a subject by an enveloped virus, includingbut not limited to prophylactic treatment and therapeutic treatment. Assuch, the terms treatment or treating include, but are not limited to:preventing an enveloped viral infection or the development of anenveloped viral infection; inhibiting the progression of an envelopedviral infection; arresting or preventing the development of an envelopedviral infection; reducing the severity of an enveloped viral infection;ameliorating or relieving symptoms associated with an enveloped viralinfection; and causing a regression of the enveloped viral infection orone or more of the symptoms associated with the enveloped viralinfection.

The phrase “enveloped viral infection” is used herein to refer to anyinfection that is caused, at least in part, or exacerbated by thereproduction and proliferation of enveloped viruses within the body of asubject, including, but not limited to: the reproduction andproliferation of enveloped DNA viruses, such as herpesviruses,poxviruses, hepadnaviruses; the reproduction and proliferation ofenveloped RNA viruses, such as flavivirus, togavirus, coronavirus,hepatitis D, orthomyxovirus, paramyxovirus, rhabdovirus, bunyavirus,filovirus; and the reproduction and proliferation of envelopedretroviruses, such as human immunodeficiency virus (HIV) andhepadnavirus. As noted herein, enveloped viruses are characterized byviral envelopes that cover the protein capsids of the viruses and aretypically comprised of portions of the host cell lipid membranes and anumber of glycoproteins that serve to identify and bind to receptorsites on the host cell membranes. It has been determined, however, thatthe administration of a polypeptide of the presently-disclosed subjectmatter (e.g., a fusion protein comprising an AH_(var) polypeptide and anantibody fragment, such as an Fc fragment) to a subject is useful in thetreatment of an enveloped viral infection, as defined herein. Inparticular, it has been determined that the polypeptides of thepresently-disclosed subject matter are capable of binding to the outerglycoproteins of the envelopes found on enveloped viruses and are ableto prevent or otherwise reduce the binding of the enveloped viruses to ahost cell. As such, in some embodiments of the therapeutic methodsdescribed herein, the enveloped viruses are selected from a humanimmunodeficiency virus (HIV), an influenza virus, a hanta virus, ahepatitis C virus, a herpes virus, a severe acute respiratory syndromecoronavirus (SARS-CoV), a metapneumovirus, a henipavirus (e.g.,Hendravirus and Nipahvirus), a flavivirus (e.g., Dengue and West Nileviruses) and a hemorrhagic fever virus (e.g., Marburg and Ebolaviruses).

For administration of a therapeutic composition as disclosed herein,conventional methods of extrapolating human dosage based on dosesadministered to a murine animal model can be carried out using theconversion factor for converting the mouse dosage to human dosage: DoseHuman per kg=Dose Mouse per kg×12 (Freireich, et al., (1966) CancerChemother Rep. 50:219-244). Drug doses can also be given in milligramsper square meter of body surface area because this method rather thanbody weight achieves a good correlation to certain metabolic andexcretionary functions. Moreover, body surface area can be used as acommon denominator for drug dosage in adults and children as well as indifferent animal species as described by Freireich, et al. (Freireich etal., (1966) Cancer Chemother Rep. 50:219-244). Briefly, to express amg/kg dose in any given species as the equivalent mg/sq m dose, multiplythe dose by the appropriate km factor. In an adult human, 100 mg/kg isequivalent to 100 mg/kg×37 kg/sq m=3700 mg/m².

Suitable methods for administering a therapeutic composition inaccordance with the methods of the present invention include, but arenot limited to, systemic administration, parenteral administration(including intravascular, intramuscular, intraarterial administration),oral delivery, topical administration, buccal delivery, rectal delivery,vaginal delivery, subcutaneous administration, intraperitonealadministration, inhalation, intratracheal installation, surgicalimplantation, transdermal delivery, local injection, and hyper-velocityinjection/bombardment. Where applicable, continuous infusion can enhancedrug accumulation at a target site (see, e.g., U.S. Pat. No. 6,180,082).In some embodiments, such as those which include a pharmaceuticalcomposition comprising a AH_(var) polypeptide of the presently-disclosedsubject matter, the pharmaceutical composition can be administeredtopically to thereby treat a viral infection.

Regardless of the route of administration, the compounds of the presentinvention are typically administered in amount effective to achieve thedesired response. As used herein, the terms “effective amount” and“therapeutically effective amount” refer to an amount of the therapeuticcomposition (e.g., a composition comprising an AH_(var) polypeptide, anda pharmaceutically vehicle, carrier, or excipient) sufficient to producea measurable biological response (e.g., a decrease in the amount of anenveloped viral infection). Actual dosage levels of active ingredientsin a therapeutic composition of the present invention can be varied soas to administer an amount of the active polypeptide(s) that iseffective to achieve the desired therapeutic response for a particularsubject and/or application. Of course, the effective amount in anyparticular case will depend upon a variety of factors including theactivity of the therapeutic composition, formulation, the route ofadministration, combination with other drugs or treatments, severity ofthe condition being treated, and the physical condition and priormedical history of the subject being treated. Preferably, a minimal doseis administered, and the dose is escalated in the absence ofdose-limiting toxicity to a minimally effective amount. Determinationand adjustment of a therapeutically effective dose, as well asevaluation of when and how to make such adjustments, are known to thoseof ordinary skill in the art.

For additional guidance regarding formulation and dose, see U.S. Pat.Nos. 5,326,902 and 5,234,933; PCT International Publication No. WO93/25521; Berkow, et al., (1997) The Merck Manual of MedicalInformation, Home ed. Merck Research Laboratories, Whitehouse Station,N.J.; Goodman, et al., (2006) Goodman & Gilman's the PharmacologicalBasis of Therapeutics, 11th ed. McGraw-Hill Health Professions Division,New York; Ebadi. (1998) CRC Desk Reference of Clinical Pharmacology. CRCPress, Boca Raton, Fla.; Katzung, (2007) Basic & Clinical Pharmacology,10th ed. Lange Medical Books/McGraw-Hill Medical Pub. Division, NewYork; Remington, et al., (1990) Remington's Pharmaceutical Sciences,18th ed. Mack Pub. Co., Easton, Pa.; Speight, et al., (1997) Avery'sDrug Treatment: A Guide to the Properties, Choice, Therapeutic Use andEconomic Value of Drugs in Disease Management, 4th ed. AdisInternational, Auckland/Philadelphia; and Duch, et al., (1998) Toxicol.Lett. 100-101:255-263, each of which are incorporated herein byreference.

As used herein, the term “subject” includes both human and animalsubjects. Thus, veterinary therapeutic uses are provided in accordancewith the presently-disclosed subject matter. As such, thepresently-disclosed subject matter provides for the treatment of mammalssuch as humans, as well as those mammals of importance due to beingendangered, such as Siberian tigers; of economic importance, such asanimals raised on farms for consumption by humans; and/or animals ofsocial importance to humans, such as animals kept as pets or in zoos.Examples of such animals include but are not limited to: carnivores suchas cats and dogs; swine, including pigs, hogs, and wild boars; ruminantsand/or ungulates such as cattle, oxen, sheep, giraffes, deer, goats,bison, and camels; and horses. Also provided is the treatment of birds,including the treatment of those kinds of birds that are endangeredand/or kept in zoos, as well as fowl, and more particularly domesticatedfowl, i.e., poultry, such as turkeys, chickens, ducks, geese, guineafowl, and the like, as they are also of economic importance to humans.Thus, also provided is the treatment of livestock, including, but notlimited to, domesticated swine, ruminants, ungulates, horses (includingrace horses), poultry, and the like.

The practice of the presently-disclosed subject matter can employ,unless otherwise indicated, conventional techniques of cell biology,cell culture, molecular biology, transgenic biology, microbiology,recombinant DNA, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature. See e.g.,Molecular Cloning A Laboratory Manual (1989), 2nd Ed., ed. by Sambrook,Fritsch and Maniatis, eds., Cold Spring Harbor Laboratory Press,Chapters 16 and 17; U.S. Pat. No. 4,683,195; DNA Cloning, Volumes I andII, Glover, ed., 1985; Oligonucleotide Synthesis, M. J. Gait, ed., 1984;Nucleic Acid Hybridization, D. Hames & S. J. Higgins, eds., 1984;Transcription and Translation, B. D. Hames & S. J. Higgins, eds., 1984;Culture Of Animal Cells, R. I. Freshney, Alan R. Liss, Inc., 1987;Immobilized Cells And Enzymes, IRL Press, 1986; Perbal (1984), APractical Guide To Molecular Cloning; See Methods In Enzymology(Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells,J. H. Miller and M. P. Calos, eds., Cold Spring Harbor Laboratory, 1987;Methods In Enzymology, Vols. 154 and 155, Wu et al., eds., AcademicPress Inc., N.Y.; Immunochemical Methods In Cell And Molecular Biology(Mayer and Walker, eds., Academic Press, London, 1987; Handbook OfExperimental Immunology, Volumes I-IV, D. M. Weir and C. C. Blackwell,eds., 1986.

The presently-disclosed subject matter is further illustrated by thefollowing specific but non-limiting examples. Some of the followingexamples may include compilations of data that are representative ofdata gathered at various times during the course of development andexperimentation related to the present invention. Additionally, certainof the following examples are prophetic, notwithstanding the numericalvalues, results and/or data referred to and contained in the examples.

EXAMPLES Example 1 Generation of Actinohivin Variant (AH_(var))Polypeptide

Due to observations of poor expression and poor solubility in aqueousbuffer solutions when actinohivin (AH) polypeptides were expressed inplants, modifications to the AH polypeptide sequence were undertaken inan attempt to increase the expression of the polypeptides in plants andto increase the solubility of the polypeptides. Without wishing to bebound by any particular theory, it was believed that one of the causesof the low expression of the AH polypeptides was due to AH's relativelyhigh isoelectric point (greater than 8). As such, an initialmodification to the amino acid sequence changed the glutamine residue atthe sixth position within the first and second carbohydrate-bindingdomains of the AH polypeptide to glutamic acid, such that the glutamicacid residues in the sixth position of the first and secondcarbohydrate-binding domains corresponded to the glutamic acid residuein the third carbohydrate binding domain of the AH polypeptide. Alsowithout wishing to be bound by any particular theory, it was furtherbelieved that the poor expression was due to the poor stability of theAH polypeptide and, as such, the first and third carbohydrate-bindingdomains were further modified such that the produced variant proteinswould include two cysteine residues that would then facilitate theformation of an intra-domain disulfide bond, such as what is found inthe second carbohydrate-binding domain. To further improve thesolubility, it was also believed that it was necessary to reduce thehydrophobicity of the protein and, to this end, the second hinge regionof AH was modified to resemble the first hinge region, as the formercontained very hydrophobic residues such as leucine and phenylalanine,whereas the latter was more charged and was thus more hydrophilic. For acomparison of the native AH polypeptide with the initially constructedAH variant polypeptide (i.e., AH_(var1) polypeptide), see, e.g., FIGS.1A and 1B.

Following the design of the modified AH polypeptide (AH_(var1); SEQ IDNO: 4), a nucleic acid sequence (SEQ ID NO: 3) encoding the modifiedpolypeptide was then synthesized and the AH_(var1) polypeptide wassubsequently expressed in Nicotiana benthamiana using the magnICONsystem (Icon Genetics Inc., Halle/Saale, Germany), where the nucleicacid sequence encoding the AH_(var1) polypeptide was cloned into apICH11599 vector, both with a signal peptide to target the AH_(var1)polypeptide to the endomembrane system (EMS) and without a signalpeptide to allow for cytosolic expression of the polypeptide. Uponanalysis of the Nicotiana benthamiana expressing these polypeptides, andas shown in FIG. 2, it was found that AH_(var1) polypeptides werehighly-expressed in the plant tissues as compared to the levels ofnative AH polypeptides that were expressed in the same tissues,indicating that the amino acid modifications improved the expression ofthe polypeptide in plant tissues.

Example 2 Anti-Human Immunodeficiency Virus Activity of ActinohivinVariant (AH_(var)) Polypeptide

To further assess the properties of the AH_(var1) polypeptide and, inparticular to assess the ability of the AH_(var1) polypeptide to displayanti-human immunodeficiency virus (HIV) activity, a syncytium formationassay was performed using HeLa/env/tat and HeLa/CD4/lacZ cell lines aspreviously described in Matoba et al. PLoS ONE, 2010. Briefly, in96-well plates, serially-diluted, clarified leaf extracts, either in PBSfor the AH_(var1) polypeptide or in 2% SDS for native AH, were mixedwith 9,000 cells/well of the two cell lines and were incubated for 18hrs at 37° C. The syncytium formation was then quantified bybeta-galactosidase activity of cell lysates. As shown in FIG. 3, uponanalysis of the results from these experiments, it was found that theAH_(var1) polypeptide (Plant mAH) was able to reduce the percentage ofsyncytiums formed, thus indicating that the AH_(var1) polypeptide isuseful for treating an enveloped virus infection.

Example 3 Generation, Expression, and Activity of Additional ActinohivinVariant (AH_(var)) Polypeptides

Based on the results obtained with the initial AH_(var1) polypeptide, 8additional AH variant polypeptide were designed and tested. Each ofthese AH variant polypeptides (AH_(var2-9) polypeptides; SEQ ID NOS:5-12) were observed to be capable of expression in plants. Additionally,each of the further variant polypeptides are shown to be capable ofreducing the percentage of syncytiums formed in an Env-CD₄ syncytiumformation assay, indicating that the further AH variant polypeptidesfound in SEQ ID NOS: 5-12 are also useful for treating an envelopedvirus infection.

Example 4 Generation and Expression of Actinohivin Variant (AH_(var))Fusion Protein

To examine whether combining the AH_(var1) polypeptide with an antibodyfragment would further improve the properties of the AH_(var1)polypeptide, an AH_(var1) polypeptide-Fc fusion protein was constructedby attaching the nucleic acid sequence encoding for the hinge and the Fcregion of a human immunoglobulin (Ig)G to the 3′ end of the AH_(var1)polypeptide-coding sequence via a flexible peptide linker(Gly-Gly-Gly-Ser; SEQ ID NO: 13) coding sequence to potentially improvethe antiviral activity of the fusion proteins (see, e.g., FIG. 4). Inaddition, a DNA sequence coding for an endomembrane system(EMS)-targeting signal peptide derived from rice alpha-amylase wasattached to the 5′ end of the AH_(var1) polypeptide-coding sequence todirect the expression of the AH_(var1) polypeptide to the plant's EMS.Without wishing to be bound by any particular theory, it was believedthat the AH_(var1) polypeptide could be produced as part of a Fc fusionprotein, as well as an Fab and scFv antibody fusion protein, due to thefinding that the AH_(var1) polypeptide was highly expressed in theapoplast via the plant cell's EMS (i.e., the endoplasmic reticulum andthe Golgi apparatus) where antibody fragments are properly assembledinto functional molecules, while, other well-knownhigh-mannose-glycan-specific anti-HIV lectins, such as griffithsin andcyanovirin-N, do not appear to be compatible with EMS-based highexpression

Once the nucleic acid sequence for the AH_(var1) polypeptide-Fc fusionprotein was designed and constructed, a “deconstructed” tobamovirusreplicon system (magnICON; Icon Genetics GmbH, Halle/Saale, Germany) wasthen used to express the AH_(var1) polypeptide-Fc fusion protein in N.benthamiana. Briefly, the AH_(var1) polypeptide-Fc fusion protein-codingDNA sequence was sub-cloned into the vector pICH38099, and the plasmidwas transferred into the Agrobacterium tumefaciens strain GV3101 byelectroporation. The bacteria were then resuspended in an infiltrationbuffer (10 mM 2-(N-morpholino)ethanesulphonic acid [MES], 10 mM MgSO₄,pH 5.5) to give an optical density at 600 nm (OD600) of 0.03. The N.benthamiana plants were grown at 27° C. and 55 to 65% humidity for 4weeks under an 18 h-light/6 h-dark cycle, and the bacteria suspensionwas subsequently infiltrated into leaves by application of a vacuum for2 min at 25 inches Hg using a vacuum pump. After infiltration, theplants were placed in the growth chamber set at the same conditionsdescribed above. At 7 days post infiltration, infected leaves were thenharvested and examined for AH_(var1) polypeptide-Fc fusion proteinexpression by SDS-PAGE (FIG. 5). The protein was detected at a predictedsize of approximately 37 kilodaltons and was expressed at a very highlevel, reaching 2 to 3 g per kg of leaf material. SDS-PAGE analysisunder non-reducing conditions (without 2-mercaptoethanol in the samplebuffer) revealed that the majority of the expressed protein forms adimer via disulfide bonding like immunoglobulins. Western blot analysisfurther confirmed that the expressed protein possessed both AH_(var1)polypeptide and Fc domains. The Nicotiana-expressed AH_(var1)polypeptide-Fc fusion protein's ability to bind to HIV gp120 was furtherdemonstrated by gp120-capture enzyme-linked immunosorbent assay(gp120-ELISA). The protein was then extracted and purified using aProtein G column for further analysis.

Without wishing to be bound by any particular theory, and based on theresults obtained with an AH_(var1) polypeptide-Fc fusion protein, it isbelieved that other AH_(var1) polypeptide-immunoglobulin fusions can bedeveloped in a similar fashion, including AH_(var1) polypeptide-Fab andAH_(var1) polypeptide-scFv fusion proteins. More specifically, it isbelieved that Fab and scFv molecules can be attached to either or boththe N- and/or C-termini of the AH_(var1) polypeptide because the crystalstructure of AH (Protein Data Bank ID: 3a07) revealed that both the N-and C-termini are exposed and located away from the putativecarbohydrate-binding pockets of the lectin.

Example 5 Anti-Human Immunodeficiency Virus Activity of ActinohivinVariant (AH_(var)) Fusion Protein

To further examine the properties of the AH_(var1) polypeptide-Fc fusionprotein, the antiviral activity of AH and the AH_(var1) polypeptide-Fcfusion protein was assessed based on a reduction in luciferase reportergene expression after infection of TZM-bl cells with HIV envelope(Env)-pseudotyped viruses. The assay was performed as described in:Matoba et al. PLoS ONE 2010 e11143, with antiviral activity beingexpressed as a percentage of neutralization, and where an averagerelative luminescence unit (RLU) obtained at each concentration of theAH_(var1) polypeptide-Fc fusion protein was compared with that of thevirus control after subtraction of background RLUs. Briefly,Env-pseudotyped viruses were prepared by co-transfection of 293T/17cells with a MN strain's env-expressing plasmid and an env-deficientHIV-1 backbone vector (pSG3ΔEnv). Samples and the virus were mixed andincubated for 1 h at 37° C., to which 10⁴ cells/well of TZM-bl cellswere added and incubated for 72 h. Luciferase activity of the celllysates was then measured using a plate reader. Upon analysis of theresults from the assay, it was observed that AH dose-dependentlyneutralized the virus with a 50% inhibitory concentration (IC₅₀) ofapproximately 0.5 μg/ml (FIG. 6; Actinohivin). The AH_(var1)polypeptide-Fc fusion protein, however, exhibited a much more potentactivity, with an IC₅₀ of less than 0.001 μg/ml (FIG. 6; AH_(var)-Fc),indicating that the fusion of the AH_(var1) polypeptide to an IgG Fcfragment enhances the anti-HIV activity of the variant polypeptide,while possibly also providing additional efficacy via variousFc-mediated biological functions, such as antibody-dependentcell-mediated cytotoxicity, complement-dependent cytotoxicity,antibody-dependent cellular phagocytosis, antibody-dependentcell-mediated virus inhibition, as well as a long serum half life uponsystemic use (see, e.g., Forthal et al., Curr Opin HIV AIDS 2009, 4;388-393; Moore et al., mAbs 2010, 2; 18-189, see also, e.g., Hansel etal., Nat Rev Drug Discov 2010; 9(4):325-38; and Moore et al., mAbs 2010,2; 18-189, describing how manipulation of Fc-mediated activities (e.g.,augmentation, reduction, or elimination) can be achieved by modifyingthe Fc sequence, each of which are incorporated herein by reference).

Throughout this document, various references are mentioned. All suchreferences are incorporated herein by reference, including thereferences set forth in the following list:

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It will be understood that various details of the presently disclosedsubject matter can be changed without departing from the scope of thesubject matter disclosed herein. Furthermore, the foregoing descriptionis for the purpose of illustration only, and not for the purpose oflimitation.

What is claimed is:
 1. An isolated polypeptide comprising an actinohivinvariant polypeptide having an amino acid sequence selected from thegroup consisting of SEQ ID NOs:4-12.
 2. The polypeptide of claim 1,wherein the actinohivin variant polypeptide comprises the sequence ofSEQ ID NO:
 4. 3. The polypeptide of claim 1, further comprising a secondpolypeptide selected from: a fragment crystallizable domain of anantibody (Fc); a fragment antigen-binding domain of an antibody (Fab);and a single chain variable fragment of an antibody (scFv), wherein theactinohivin variant polypeptide and the second polypeptide comprise afusion protein.
 4. The polypeptide of claim 3, wherein the antibody is amonoclonal antibody.
 5. The polypeptide of claim 3, wherein the secondpolypeptide is Fab.
 6. The polypeptide of claim 3, wherein the secondpolypeptide is scFv.
 7. The polypeptide of claim 3, wherein the secondpolypeptide is Fc.
 8. The polypeptide of claim 3, wherein thepolypeptide further comprises a peptide linker for connecting theactinohivin variant polypeptide to the second polypeptide.
 9. Thepolypeptide of claim 3, wherein the peptide linker comprises an aminoacid sequence of SEQ ID NO:
 13. 10. The polypeptide of claim 1, whereinthe actinohivin variant polypeptide further comprises an endomembranesystem (EMS)-targeting signal peptide.
 11. The polypeptide of claim 10,wherein the EMS-targeting signal peptide comprises an amino acidsequence of SEQ ID NO:
 15. 12. A polypeptide comprising an actinohivinvariant polypeptide of claim 1 and a second polypeptide selected fromthe group consisting of: a fragment of antigen-binding domain of anantibody (Fab); and a single chain variable fragment of an antibody(scFv).
 13. An isolated nucleic acid molecule, comprising a sequencethat encodes an actinohivin variant polypeptide having an amino acidsequence selected from SEQ ID NOS: 4-12.
 14. The isolated nucleic acidmolecule of claim 13, wherein the nucleic acid molecule comprises thesequence of SEQ ID NO:
 3. 15. A vector, comprising the nucleic acidmolecule of claim
 13. 16. The vector of claim 15, wherein the isolatednucleic acid is operably linked to an expression control sequence.
 17. Aplant cell transfected with the vector of claim 15, or a progeny of theplant cell, wherein the plant cell or the progeny thereof expresses theactinohivin variant polypeptide.
 18. The plant cell of claim 17, whereinthe plant cell is a Nicotiana benthamiana plant cell.
 19. A compositioncomprising the polypeptide of claim 1 and a pharmaceutically-acceptablevehicle, carrier, or excipient.